1. Field of the Invention
The present invention relates generally to the fields of cancer treatment and radiation therapy. More particularly, it concerns a combination of low doses of the protein kinase C (PKC) inhibitor chelerythrine with low doses of ionizing radiation to produce unexpectedly effective killing of radioresistant tumor cells.
2. Description of Related Art
Certain cancer treatment methods, including radiation therapy, involve damaging the DNA of the cancer cell. The cellular response to DNA damage includes activation of DNA repair, cell cycle arrest and lethality (Hall, 1988). Ionizing radiation (IR; x-irradiation) mediated killing of mammalian cells is frequently characterized by loss of reproductive integrity after one or more cell divisions (Chang and Little, 1992a, 1991, 1992b; Bussink et al., 1996). The induction of DNA double-strand breaks results in lethal chromosomal aberrations that include deletions, dicentrics, rings, and anaphase bridges (Hall, 1994). The morphological characteristics of cells dying a mitotic death following IR exposure include, as in necrotic death, multi-nucleated giant cells, cell-cell fusions (Hall, 1994), as well as the loss of membrane integrity (Quintans et al., 1994; Maity et al., 1994; Harmon and Allan, 1988; Radford and Murphy, 1994). In contrast to necrotic death, morphological characteristics of apoptosis induced by IR (Quintans et al., 1994; Maity et al., 1994; Harmon and Allan, 1988; Radford and Murphy, 1994) include activation of a genetic program that may be initiated by cytoplasmic or nuclear events which results in cytoplasmic blebbing, chromatin condensation, and DNA fragmentation (Jacobson et al., 1994; Raff et al., 1994).
Studies in tumor systems suggest that increasing the fraction of tumor cells undergoing apoptosis enhances tumor regression and tumor cures by IR (Meyn et al., 1993; 1994; 1995; Martin and Green, 1994; Indap and Rao, 1995; Dewey et al., 1995; Lowe et al., 1993b; Stephens et al., 1991; 1993). Agents which damage DNA, interfere with DNA repair, or alter cell-cycle checkpoints have been employed in human studies to modify the radiation tumor response with limited clinical success (Hall, 1988; Vokes and Weichselbaum, 1990; Rosenthal et al., 1995). Probably in part because loss of the apoptotic response to x-irradiation has been linked to a radiation resistant phenotype. Tumor radioresistance may occur through activation of DNA repair genes and cell cycle checkpoints (Maity et al., 1994; Sanchez and Elledge, 1995; Szumiel, 1994; Park, 1995; Shen et al., 1996; McKenna et al., 1991). For example, loss of p53 function is associated with a radioresistant phenotype as a consequence of a diminished ability to undergo apoptosis (Boise et al., 1995) and is often associated with treatment resistance and tumor relapse (Lowe et al., 1993a; 1993b; 1994). An increased ratio of anti-apoptotic to pro-apoptotic proteins also decreases IR-induced apoptosis.
Sphingomyelinase activation and subsequent ceramide production has been demonstrated to precede x-ray-induced apoptosis in several cell types (Quintans et al., 1994; Haimovitz-Friedman et al., 1994; Verheij et al., 1996; Kolesnick, 1994; Jarvis and Kolesnick, 1996; Santana et al., 1996). The loss of ceramide production following x-rays has recently been shown to confer a radioresistant phenotype in cells derived from acidic sphingomyelinase knockout mice and in tumor cells selected for a defect in neutral sphingomyelinase production. In addition, chelerythrine chloride (Herbert et al., 1990) and calphostin C (Kobayashi et al., 1989b), inhibitors of PKC isoforms, induce apoptosis and ceramide production through the activation of a neutral sphingomyelinase (Chmura et al., 1996a; Chmura et al., 1996b).
Ionizing radiation induces PKC and protein tyrosine kinase activities (Hallahan et al., 1990; Uckun et al., 1995). However, the specific kinases responsible for these activities and their substrates are not completely understood. PKC activation is functionally related to gene induction following exposure of mammalian cells to IR (Young et al., 1994; Kondratyev et al., 1996; Hallahan et al., 1995; Hallahan et al., 1994). The protein kinase C family of serine/threonine kinases is comprised of at least 13 related isoforms (Magnuson et al., 1994) with differing sensitivity to calcium and lipid activators. PKC activation is also reported to limit the production of ceramide from the hydrolysis of sphingomyelin and rescue cells from IR mediated apoptosis (Kolesnick et al., 1994; Haimovitz-Friedman et al., 1994).
Little information is available concerning the relationship between PKC inhibitors and the induction of programmed cell death in human tumor cells, and the results described in existing reports are inconsistent. For example, the potent, but nonspecific, PKC inhibitor staurosporine has been reported both to antagonize (Cotter et al., 1992) and to initiate apoptosis in HL-60 cells (Bertrand et al., 1993); similarly conflicting reports of the action of the inhibitor H7 have also appeared (Ojeda et al., 1990; Forbes et al., 1992). Detailed comparisons of the concentration-response relationships of different PKC inhibitors in the modulation of apoptosis are generally lacking. Jarvis et al. (1994) demonstrate that, while the effects of these agents are variable and highly dependent upon concentration, transient exposure of HL-60 cells to a subset of PKC inhibitors, in particular chelerythrine, unambiguously induces apoptotic DNA fragmentation and cell death in HL-60 cells and that acute (i.e., 6-h) exposure to chelerythrine is sufficient to induce apoptosis in the human myeloid leukemia cell line HL-60. In addition, in vitro treatment of certain cells with inhibitors of PKC and other serine-threonine kinases increases IR mediated killing through undefined mechanisms (Hallahan et al., 1992).
Recent investigations indicate that signaling events following cellular exposure to tumor necrosis factor alpha (TNF.alpha.), Fas ligand, IgM cross-linking, irradiation and other DNA damaging agents may trigger apoptosis via the hydrolysis of membrane sphingomyelin generating ceramide (Quintans et al., 1994; Nagata and Golstein, 1995; Dressier et al., 1992). Activation of protein kinase C by phorbol esters or growth factors opposes ceramide induced apoptosis and indirect evidence suggests that PKC activation may limit ceramide production (Fuks et al., 1994; Haimovitz-Friedman et al., 1994a; Haimovitz-Friedman et al., 1994b). One potential action of ceramide and its metabolite sphingosine is to prevent activation of specific PKC isoforms (Chmura et al., 1996b; Jones and Murray, 1995; Kolesnick, 1989; Ohta et al., 1994). Taken together, these studies suggest that PKC activation may oppose the actions of ceramide production in the apoptotic pathway.
Recent studies investigating mechanisms of radiation-mediated apoptosis demonstrate that the production of the lipid second messenger ceramide from sphingomyelin hydrolysis immediately following x-rays contributes to the apoptotic response (Kolesnick et al., 1994; Haimovitz-Friedman et al., 1994b; Verheij et al., 1996). Two forms of sphingomyelinase have been implicated in the generation of ceramide following x-rays. Involvement of the Mg.sup.++ dependent neutral sphingomyelinase was first suggested as the source of ceramide generation at the membrane following ionizing radiation in endothelial cells (Haimovitz-Friedman et al., 1994). Recently Santana et al. demonstrated that tissues and cells from acidic sphingomyelinase knockout mice are more resistant to apoptosis following ionizing radiation (Santana et al., 1996). This study implicated acidic sphingomyelinase as a component of the apoptotic response in some tissues.
Much of the signaling interaction of PKC isoforms within tumor cells remains to be elucidated. It is clear that PKC pathways play a key role in the control of apoptosis by tumor cells. It is also evident that these pathways are altered in radioresistant tumor cells which results in an increased difficulty in treating these types of tumors. There is a present need to develop new and improved treatments which overcome the resistance to apoptosis of radioresistant tumors.